In the field of modern cell biology, microbiology, and especially molecular biology, it is an important research subject to find what molecules a cell and its organelles are made of, how and what the molecules are made in which organelles, where the molecules are transported to, how their molecular structures change, and how they are decomposed in a cell and its organelles. The movement and growth of cells are the essential factors governing the normal physiological process of cells. If the movement and growth of cells are restricted, negative results such as tumor formations are induced.
Meanwhile, it is important to know the three-dimensional distribution and change of the molecules or elements constituting a cell in order to understand the cellular structure and the functions of individual cellular organelles. A representative method to find the three-dimensional distribution of the molecules and organelles constituting a cell is a fluorescent imaging technique.
The fluorescent imaging technique allows one to acquire fluorescent images after attaching the specific molecules of interest with florescent markers or after genetically engineering the specific molecules to express fluorescence, and to confirm the three-dimensional distribution of specific molecules. As an exemplary apparatus with the fluorescent imaging technique applied is a confocal laser microscope. However, the fluorescent technique has shortcomings that cells are imaged while in an unnatural state or an artificially engineered state where genetically engineered fluorescence is expressed or foreign fluorescence materials are introduced into the cell. In addition, in order to be able to observe specific molecules via the fluorescent imaging technique, the concentration of the molecules in the cell should be higher than a certain threshold level. Even after cells are genetically engineered to express fluorescence or after the fluorescent markers are attached, the molecules with low concentrations cannot be imaged. Moreover, because the fluorescence image comes from the fluorescent markers, one cannot know for sure whether the markers are attached to the specific molecules of interest.
If the concentration of the specific molecule of interest is low, one can increase the concentrations using the methods such as to over-stimulate the cells to overexpress the molecules of interest or to amplify the molecules in the case of DNA and RNA molecules. There is a generally used method in that over-stimulated and processed cells and control group cells are cultured at the same time to increase the number of the cells, then the cells are separated, lysed (cell lysis), centrifuged, and certain molecules of interest in the cells are examined to see their increase or decrease using gel electrophoresis or mass spectroscopy. But these methods take a long time and need expensive reagents. Besides, it is difficult to continuously observe the cells because the cells are fixed and dead. Moreover, the amount of expression of the molecules of interest may differ because some molecules have to be over-stimulated in order to measure.
As the cell imaging methods beside the said optical method and the said biochemical method, there are other methods to research the structures of cells and their organelles and the molecules constituting the cells and the organelles such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM), and scanning tunneling microscopy (STM).
Scanning electron microscopy is used to observe specimens by detecting the secondary electrons or backscattered electrons that are the most probable among the various signals generated from specimens when an electron beam scans over the sample surface. However, as for the electron scanning microscopy, it is difficult to image molecules, and it has a shortcoming that specimens have to be coated with high atomic number metallic materials after freeze drying.
Transmission electron microscopy is used to observe samples by enlarging with electric lenses the parallel electron beam after passing through the samples. Molecules can be imaged by the transmission electron microscopy, but it has shortcomings that it takes a long time to image and analyze a specimen and that the specimen has to be frozen and cut into thin slices.
An atomic force microscope is a microscope in that a pyramid shaped probe contacts the surface of a sample and scans two dimensionally. As for the atomic force microscope, inorganic or metallic specimens can be imaged. However, there is a problem of rapidly degrading resolving power when imaging cells containing water with 75˜80% of the volume and the cells immersed in a culturing media. Also, instead of the excellent resolving power, the imaging area is very small, and the reliability of the cell images may be low because the living cells may change their shapes voluntarily or due to the effect of the probe.
A scanning tunneling microscope, a kind of scanning probe microscope, analyzes the surface configurations of a sample utilizing tunneling currents. In the case of the scanning tunneling microscope imaging with atomic resolution is possible, but imaging is impossible for the cells containing a lot of moisture because a scanning tunneling microscope is operable in a high vacuum.
Throughout the present description, many published papers and patents are referenced, and their citations are presented. The cited papers and the disclosed content of the patents, inserted as the reference in the present description statement, explain more clearly the level of the technology and the content of the present invention in the technological area to which the present invention belongs.